LOCALIZATION OF TAU AND OTHER PROTEINS OF ISOLATED MARGINAL BANDS

To determine which proteins were associated with and intrinsic to the marginal band (MB) of microtubules (MTs), we studied protein component s of MBs isolated from nucleated erythrocytes by differential detergen t solubilization of the membrane skeleton (MS). MBs isolated from dogf ish erythrocytes contained major proteins in the tubulin M(r) range. A high molecular weight protein of approximately 290 kD that bound anti body to syncolin and to heat-stable brain MAPs was present in the whol e cytoskeleton. However, most of it was solubilized by the MB isolatio n medium, together with the MS. Dogfish erythrocyte cytoskeletons and isolated MBs were examined with polyclonal and monoclonal antibodies a gainst mammalian brain tau and chicken erythrocyte tau. As shown by im munofluorescence and immunoblotting, these antibodies bound to protein s in the 50 to 67 kD range, located along the length of isolated MBs. Two-dimensional SDS-PAGE revealed isolated MB proteins of pI approxima tely 6.8 in the same molecular weight range, as well as alpha- and bet a-tubulin with pI approximately 5.4. Subtilisin or high-salt treatment of isolated MBs resulted in unbundling of MTs, indicating involvement of MAPs. MBs isolated from chicken erythrocyte cytoskeletons also con tained tau as shown by anti-mammalian brain tau immunofluorescence. Bo th chicken and dogfish isolated MBs also bound phalloidin, but the bin ding was usually discontinuous and, for any given MB, matched the patt ern of anti-syncolin binding. Both syncolin and F-actin were part of t he MS remnant remaining after MT disassembly, supporting their assignm ent to a specialized MS region at the MB/MS interface. In contrast, ta u protein appears to be intrinsic to the MB, where it may have an MT s tabilizing and bundling function. (C) 1994 Wiley-Liss, Inc.